Aldo Orlacchio

Identification of Hematopoietic Stem Cell–Specific miRNAs Enables Gene Therapy of Globoid Cell Leukodystrophy

Hematopoietic stem cell–specific microRNAs allow regulation of therapeutic transgene expression and enable effective gene therapy of globoid cell leukodystrophy. Scratching the Surface of the Holy Grail In Monty Python and the Holy Grail, when King Arthur cuts off one of the arms of the Black Knight, he claims it is only a scratch. Similarly, gene therapy—the insertion of genes into cells to reverse a condition or repair a biological process—has been heralded as a Holy Grail for the treatment of genetic diseases for nearly 40 years. Yet, the complications of gene therapy, including immune responses to the viral vector and cancers that result from insertional mutagenesis, are more comparable to a severed arm than a surface wound. However, researchers with the resiliency of the Black Knight have presided over recent successes, most notably in metastatic melanoma and immune cells, and have reignited the quest for gene therapy solutions to otherwise untreatable diseases. Gentner et al. build on these successes by identifying new microRNAs that can restrict gene therapy vectors to particular immune cell types and thus be used to safely treat globoid cell leukodystrophy (also known as Krabbe disease). Globoid cell leukodystrophy is a rare metabolic disorder caused by a mutation in a lysosomal enzyme called galactocerebrosidase (GALC). In patients who carry the mutation in both copies of the GALC gene, unmetabolized lipids accumulate in myelin-secreting glial cells, rendering them unable to produce the myelin sheath that normally wraps and protects nerves. This aberration results in severe and often fatal degeneration of motor skills. Bone marrow transplantation has been shown to benefit these patients if the disease is caught early enough. Genetic manipulation of the hematopoietic stem and progenitor cells (HSPCs) found in bone marrow may improve this therapy; however, high-level GALC expression in HSPCs, but not in more differentiated immune cells, is toxic. To address this issue, Gentner et al. identified miRNAs—short RNA sequences that often silence gene expression—that were specifically expressed in HSPCs but not in more differentiated cells. They then used these miRNAs in a GALC/HSPC gene therapy system to suppress GALC function in HSPCs upon transfer into a mouse model of globoid cell leukodystrophy. As these cells matured, amounts of HSPC-specific miRNA decreased and GALC expression increased. This approach protected the HSPCs from GALC toxicity, but allowed for successful gene therapy of the disease. In addition, these hematopoietic stem cell–specific miRNAs could be used as simple markers with which to isolate HSPCs for study and transplantation. This work thus provides a basis for improvements in HSPC-mediated gene therapy and may offer globoid cell leukodystrophy patients a new therapeutic option that resembles a scratch more than a chop. Globoid cell leukodystrophy (GLD; also known as Krabbe disease) is an invariably fatal lysosomal storage disorder caused by mutations in the galactocerebrosidase (GALC) gene. Hematopoietic stem cell (HSC)–based gene therapy is being explored for GLD; however, we found that forced GALC expression was toxic to HSCs and early progenitors, highlighting the need for improved regulation of vector expression. We used a genetic reporter strategy based on lentiviral vectors to detect microRNA activity in hematopoietic cells at single-cell resolution. We report that miR-126 and miR-130a were expressed in HSCs and early progenitors from both mice and humans, but not in differentiated progeny. Moreover, repopulating HSCs could be purified solely on the basis of miRNA expression, providing a new method relevant for human HSC isolation. By incorporating miR-126 target sequences into a GALC-expressing vector, we suppressed GALC expression in HSCs while maintaining robust expression in mature hematopoietic cells. This approach protected HSCs from GALC toxicity and allowed successful treatment of a mouse GLD model, providing a rationale to explore HSC-based gene therapy for GLD.

MicroRNA Implications across Neurodevelopment and Neuropathology

MicroRNAs (miRNAs) have rapidly emerged as biologically important mediators of posttranscriptional and epigenetic regulation in both plants and animals. miRNAs function through a variety of mechanisms including mRNA degradation and translational repression; additionally, miRNAs may guide gene expression by serving as transcription factors. miRNAs are highly expressed in human brain. Tissue and cell type-specific enrichments of certain miRNAs within the nervous system argue for a biological significance during neurodevelopmental stages. On the other hand, a large number of studies have reported links between alterations of miRNA homeostasis and pathologic conditions such as cancer, heart diseases, and neurodegeneration. Thus, profiles of distinct or aberrant miRNA signatures have most recently surged as one of the most fascinating interests in current biology. Here, the most recent insights into the involvement of miRNAs in the biology of the nervous system and the occurrence of neuropathological disorders are reviewed and discussed.

Stem cell-biomaterial interactions for regenerative medicine

The synergism of stem cell biology and biomaterial technology promises to have a profound impact on stem-cell-based clinical applications for tissue regeneration. Biomaterials development is rapidly advancing to display properties that, in a precise and physiological fashion, could drive stem-cell fate both in vitro and in vivo. Thus, the design of novel materials is trying to recapitulate the molecular events involved in the production, clearance and interaction of molecules within tissue in pathologic conditions and regeneration of tissue/organs. In this review we will report on the challenges behind translating stem cell biology and biomaterial innovations into novel clinical therapeutic applications for tissue and organ replacements (graphical abstract).

Intrinsic cell memory reinforces myogenic commitment of pericyte-derived iPSCs†

Mesoangioblasts (MABs) are a subset of muscle‐derived pericytes able to restore dystrophic phenotype in mice and dogs. However, their lifespan is limited and they undergo senescence after 25–30 population doublings. Recently, induced pluripotent stem cells (iPSCs) generated from reprogrammed fibroblasts have been demonstrated to have in vitro and in vivo myogenic potential when sorted for the SM/C‐2.6 antigen. Furthermore, chimeric mice from mdx‐iPSCs (DYS‐HAC) cells showed tissue‐specific expression of dystrophin. Nevertheless, myogenic differentiation protocols and the potential of iPSCs generated from different cell sources still present unanswered questions. Here we show that iPSCs generated from prospectively sorted MABs (MAB‐iPSCs) are pluripotent as fibroblast‐derived iPSCs (f‐iPSCs). However, both teratoma formation and genetic cell manipulation assays identify a durable epigenetic memory in MAB‐iPSCs, resulting in stronger myogenic commitment. Striated muscle tissue accounts for up to 70% of MAB‐iPSC teratomas. Moreover, transfection with Pax3 and Pax7 induces a more robust myogenic differentiation in MAB‐iPSCs than in f‐iPSCs. A larger amount of CD56+ progenitors can be sorted from the MAB‐iPSCs differentiating pool and, after transplantation into αsg‐KO mice, can efficiently participate to skeletal muscle regeneration and restore αsg expression. Our data strongly suggest that iPSCs are a heterogeneous population and, when generated from myogenic adult stem cells, they exhibit a stronger commitment, paving the way for creating custom‐made cell protocols for muscular dystrophies. Copyright

Lysosomal hydrolases in cerebrospinal fluid from subjects with Parkinson's disease

Recent studies have shown a genetic association between glucocerebrosidase deficiencies and Parkinsons disease (PD). To further explore this issue the activity of β‐glucocerebrosidase and the activities of other lysosomal enzymes, α‐mannosidase, β‐mannosidase, β‐hexosaminidase, and β‐galactosidase have been evaluated in the cerebrospinal fluid (CSF) of PD patients. The activities of α‐mannosidase, β‐mannosidase, β‐glucocerebrosidase, and β‐hexosaminidase were substantially decreased in the CSF of PD patients, while levels of β‐galactosidase were essentially identical to controls. This study indicates that in PD several lysosomal hydrolases have decreased activities, further supporting a possible link between pathophysiological mechanisms underlying PD and lysosomal hydrolases.

A calcium requirement for the phosphatidylinositol response following activation of presynaptic muscarinic receptors.

Abstract The labelling in vitro by [32P]phosphate of phosphatidic acid and phosphatidylinositol in synaptosomes from guinea-pig brain was studied. Acetylcholine increased the labelling and evidence is provided that pre-synaptic muscarinic receptors are involved. The increase was not seen in the presence of EGTA. Experiments with various calcium buffers indicated that concentrations of greater than 10−7 M free Ca2+ are required in the incubation medium for this phospholipid effect. A similar muscarinic effect in parotid gland is unaffected by EGTA. It is suggested that, in the parotid, postsynaptic receptors mediate increased phosphatidylinositol labelling and increased availability of Ca2+ for stimulus-secretion coupling. Presynaptic receptors similarly mediate increased labelling, which differs in being sensitive to EGTA and associated with decreased availability of Ca2+.

Widespread enzymatic correction of CNS tissues by a single intracerebral injection of therapeutic lentiviral vector in leukodystrophy mouse models

Leukodystrophies are rare diseases caused by defects in the genes coding for lysosomal enzymes that degrade several glycosphingolipids. Gene therapy for leukodystrophies requires efficient distribution of the missing enzymes in CNS tissues to prevent demyelination and neurodegeneration. In this work, we targeted the external capsule (EC), a white matter region enriched in neuronal projections, with the aim of obtaining maximal protein distribution from a single injection site. We used bidirectional (bd) lentiviral vectors (LV) (bdLV) to ensure coordinate expression of a therapeutic gene (beta-galactocerebrosidase, GALC; arylsulfatase A, ARSA) and of a reporter gene, thus monitoring simultaneously transgene distribution and enzyme reconstitution. A single EC injection of bdLV.GALC in early symptomatic twitcher mice (a murine model of globoid cell leukodystrophy) resulted in rapid and robust expression of a functional GALC protein in the telencephalon, cerebellum, brainstem and spinal cord. This led to global rescue of enzymatic activity, significant reduction of tissue storage and decrease of activated astroglia and microglia. Widespread protein distribution and complete metabolic correction were also observed after EC injection of bdLV.ARSA in a mouse model of metachromatic leukodystrophy. Our data indicated axonal transport, distribution through cerebrospinal fluid flow and cross-correction as the mechanisms contributing to widespread bioavailability of GALC and ARSA proteins in CNS tissues. LV-mediated gene delivery of lysosomal enzymes by targeting highly interconnected CNS regions is a potentially effective strategy that, combined with a treatment able to target the PNS and peripheral organs, may provide significant therapeutic benefit to patients affected by leukodystrophies.

Neural Stem Cell Gene Therapy Ameliorates Pathology and Function in a Mouse Model of Globoid Cell Leukodystrophy

Murine neural stem cells (mNSCs), either naive or genetically modified to express supranormal levels of β‐galactocerebrosidase (GALC), were transplanted into the brain of Twitcher mice, a murine model of globoid cell leukodystrophy, a severe sphingolipidosis. Cells engrafted long‐term into the host cytoarchitecture, producing functional GALC. Levels of enzyme activity in brain and spinal cord tissues were enhanced when GALC‐overexpressing NSC were used. Enzymatic correction correlated with reduced tissue storage, decreased activation of astroglia and microglia, delayed onset of symptoms, and longer lifespan. Mechanisms underlying the therapeutic effect of mNSC included widespread enzyme distribution, cross‐correction of host cells, anti‐inflammatory activity, and neuroprotection. Similar cell engraftment and metabolic correction were reproduced using human NSC. Thus, NSC gene therapy rapidly reconstitutes sustained and long‐lasting enzyme activity in central nervous system tissues. Combining this approach with treatments targeting the systemic disease associated with leukodystrophies may provide significant therapeutic benefit. STEM CELLS 2011;29:1559–1571

Cathepsin D expression is decreased in Alzheimer's disease fibroblasts

Cathepsin D (CTSD), a protease detectable in different cell types whose primary function is to degrade proteins by bulk proteolysis in lysosomes, has been suggested to be involved in Alzheimers disease (AD). In fact, there is increasing evidence that disturbance of the normal balance and localization of cathepsins may contribute to neurodegeneration in AD [Nakanishi H. Neuronal and microglial cathepsins in aging and age-related diseases. Aging Res Rev 2003; 2(4):367-81]. Here, we provide evidence of an altered balance of CTSD in skin fibroblasts from patients affected either by sporadic or familial forms of AD. In particular, we demonstrate that CTSD is down regulated at both transcriptional and translational level and its processing is altered in AD fibroblasts. The oncogene Ras is involved in the regulation of CTSD, as high expression level of the constitutively active form of Ras in normal or AD fibroblasts induces CTSD down-regulation. p38 MAPK signalling pathway also appears to down-modulate CTSD level. Overall results reinforce the hypothesis that a lysosomal impairment may be involved in AD pathogenesis and can be detected not only in the CNS but also at a peripheral level.

Identification of plasma membrane associated mature β‐hexosaminidase A, active towards GM2 ganglioside, in human fibroblasts

Mature β‐hexosaminidase A has been found associated to the external leaflet of plasma membrane of cultured fibroblasts. The plasma membrane association of β‐hexosaminidase A has been directly determined by cell surface biotinylation followed by affinity chromatography purification of the biotinylated proteins, and by immunocytochemistry. The immunological and biochemical characterization of biotinylated β‐hexosaminidase A revealed that the plasma membrane associated enzyme is fully processed, suggesting its lysosomal origin.